AllPrep DNA/RNA Micro Kit

For simultaneous purification of DNA and RNA from small cell and tissue samples

High-quality DNA and RNA from the same sample

Maximal yields of DNA and RNA from precious samples

Rapid purification with short, streamlined protocol

Ready-to-use DNA and RNA for any downstream analysis

The AllPrep DNA/RNA Micro Kit allows the simultaneous purification of genomic DNA and total RNA from the same biological sample. DNA is purified using the novel AllPrep DNA spin column. RNA is purified from the AllPrep column flow-through using an RNeasy MinElute spin column. The kit is compatible with up to 5 x 105 cells or 5 mg tissue. It can be automated on the QIAcube.

Genomic DNA and total RNA were purified from 10-fold serial dilutions of rat kidney homogenate using the AllPrep DNA/RNA Micro Kit; at each dilution, DNA was purified from duplicates, and RNA was purified from 4 replicates. [A]Real-time PCR analysis of the Pgk1 gene and [B] real-time RT-PCR analysis of the Jun transcript showed reliable quantification over a wide dynamic range, from 5 mg down to 50 ng tissue.

Performance

Both cultured cells and easy-to-lyse tissues can be processed with the AllPrep DNA/RNA Micro Kit (see table). Genomic DNA is purified using the novel AllPrep DNA spin column, and total RNA is purified using an RNeasy MinElute spin column. Total RNA with Agilent RIN values of close to 10 is routinely obtained from cultured cells. The purified genomic DNA has an average length of 15–30 kb depending on homogenization conditions (see figure "Simultaneous purification of total RNA and genomic DNA"). Genomic DNA and total RNA show reliable quantification over a wide dynamic range (see figure "Wide dynamic range").

Optimal yields of DNA and RNA from tissues

Tissue (5 mg)

DNA yield (µg)*

RNA yield (µg)*

Heart

3.89

2.00

Brain

2.60

3.08

Kidney

11.33

12.70

Liver

32.40

16.02

Lung

9.65

24.63

Spleen

20.50

43.51

Thymus

34.90

56.89

Intestine

26.71

27.38

* DNA and RNA were purified from two 5 mg samples of each tissue, and the average yields are shown.

Principle

The AllPrep DNA/RNA Micro Kit is designed for purifying both genomic DNA and total RNA from a single cell or tissue sample, and is highly suited for small samples such as laser-microdissected cryosections and fine-needle aspirates. Since there is no need to divide the sample into two for separate purification procedures, maximum yields of DNA and RNA can be achieved. The purified DNA and RNA are eluted separately and ready to use in any downstream application.

Procedure

A simple workflow allows the purification of high-quality DNA and RNA from the same sample in as little as 40 minutes (see flowchart "AllPrep DNA/RNA procedure"). Both cultured cells and easy-to-lyse tissues can be processed. Genomic DNA is purified using the novel AllPrep DNA spin column, and total RNA is purified using an RNeasy MinElute spin column.

When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the AllPrep DNA/RNA Micro Kit), excessive foaming may occur. This foaming is substantially reduced by adding Reagent DX (supplied separately) to Buffer RLT Plus at a final concentration of 0.5% (v/v) before starting disruption and homogenization. Reagent DX has been carefully tested with the kit, and has no effect on RNA purity or on downstream applications such as real-time RT-PCR. Buffer RLT Plus containing Reagent DX can be stored at room temperature (15–25ºC) for at least 9 months.

A supplementary protocol allows optional purification of protein. For experiments with larger samples, the AllPrep DNA/RNA/Protein Mini Kit can be used to simultaneously purify DNA, RNA, and protein from the same cell or tissue sample.

Applications

The purified genomic DNA has an average length of 15–30 kb, depending on homogenization conditions, and is suited for any application, including Southern-, dot-, and slot-blot analyses; and PCR and multiplex PCR.

The purified total RNA can be used in any application, such as RT-PCR and real-time RT-PCR; differential display; cDNA synthesis; northern-, dot-, and slot-blot analyses; and microarrays.

A supplementary protocol allows optional purification of protein. The purified protein is denatured and can be used in applications such as SDS-PAGE, western blotting, and 2D gel electrophoresis.